Molecular and Cellular Pathobiology Distinct Functions of Epidermal and Myeloid-Derived VEGF-A in Skin Tumorigenesis Mediated by HPV8 Xiaolei Ding 1 , Tina Lucas 1 , Gian P. Marcuzzi 2 , Herbert Pfister 2 , and Sabine A. Eming 1,3,4 Abstract Beta human papillomaviruses (HPV) have been suspected to be carcinogenic in nonmelanoma skin cancers (NMSC), but the basis for potential viral contributions to these cancers is poorly under- stood. In particular, it is unresolved how HPV-infected keratino- cytes escape cell-cycle control and whether their cross-talk with immune cells is critical for tumorigenesis. In nonviral preclinical models, the angiogenic cytokine VEGF-A has been identified as a critical regulator of NMSC. In this study, we dissected the contri- bution of epidermal versus myeloid cell–derived VEGF-A in HPV- mediated skin cancer by interbreeding an HPV8 transgenic mouse model with a conditional disruption of VEGF-A restricted to either epidermal or myeloid cells. Although only epidermal- derived VEGF-A was essential for initiation of skin tumor devel- opment, both spontaneously and UV-light triggered, both epi- dermal and myeloid cell–derived VEGF-A contributed to regen- eration-induced tumorigenesis upon HPV8 overexpression, partly not only through a paracrine effect on endothelial cells, but also most probably through an additional autocrine effect on epider- mal cells. Our findings offer new mechanistic insights into distinct functions of epidermal versus myeloid cell–derived VEGF-A dur- ing HPV-mediated tumorigenesis, with possible implications for preventing this disease. Cancer Res; 75(2); 330–43. Ó2014 AACR. Introduction Human papillomaviruses (HPV) belong to a family of small DNA viruses that can infect the epithelia of skin and mucosa. Viral persistence in the epithelium can lead to the development of benign or malignant proliferative lesions. To date, over 170 different types of HPVs have been identified (1). The causal relationship between a-genus HPV infection of mucosa (e.g., HPV16) and the development of squamous cell carcino- ma (SCC) of the genital tract is well established (2). HPV subtypes of the b-genus are suspected of playing a role in nonmelanoma skin cancer (NMSC), but their direct causal role in this process has been difficult to establish using epidemio- logic approaches due to the ubiquitous prevalence of the viruses in the general population and their absence in some cancers (3–5). In 2009, the International Agency for Research on Cancer classified HPV5 and 8 as "possibly carcinogenic" in patients with the rare inherited skin disease epidermodysplasia verruciformis (EV; ref. 6). Patients with EV are characterized by the life-long occurrence of multiple flat warts and macular lesions and develop SCC later in life, mostly in sun-exposed skin areas (7, 8). The mechanisms of HPV-mediated skin tumor development are still under debate. In contrast with mucosal high-risk HPV types (e.g., HPV16), the primary oncoproteins E6 and E7 of HPV5 and HPV8 do not or only weakly interfere directly with the central regulators of cell cycle and apoptosis p53 and pRB (3). Some activities of HPV5 and 8 E6 clearly act to enhance the carcinogenic potential of sun exposure. The E6 proteins of several beta pap- illomavirus types, including HPV5 and 8, target the proapoptotic protein Bak for degradation and thus prevent UV-induced apo- ptosis (9). They furthermore bind to p300, resulting in inhibition of p53-induced apoptosis (10), reduction of ATR mRNA and protein levels, and finally delayed repair of UV-damaged DNA (11). These activities may result in chromosomal instability particularly in the context of sun exposure and may eventually contribute to cancer development. Independent oncogenic effects may be expected from E6–MAML1 interactions, which repress the Notch signaling pathway, which has tumor suppressor function in the skin (12). HPV8 E7-induced upregulation of the MT1 matrix metalloproteinase may explain invasion of E7-positive keratino- cytes into the dermis of organotypic keratinocyte cultures (13). To gain insight into the molecular mechanisms underlying HPV8-mediated skin tumor development in vivo, we previously developed HPV8 transgenic mouse models that recapitulate the HPV8-induced SCC pathology and have been proven to be a valuable in vivo model to unravel the molecular pathology of HPV- induced skin cancer (14–18). Transgenic expression of the com- plete early genome region (CER) of HPV8 under the human keratin14 (K14) promoter in mice (FVB/N background) is suffi- cient to induce multifocal or single premalignant skin papillomas with varying degrees of epidermal dysplasia in over 90% of the mice within the first year of age (14). SCCs developed in 1 Department of Dermatology, University of Cologne, Cologne, Germany. 2 Institute of Virology, University of Cologne, Cologne, Ger- many. 3 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany. 4 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). X. Ding and T. Lucas contributed equally to this article. Corresponding Author: Sabine A. Eming, Department of Dermatology, Univer- sity of Cologne, Kerpener Street 62, 50937 Cologne, Germany. Phone: 49-221- 4783196; Fax: 49-221-4785949; E-mail: sabine.eming@uni-koeln.de doi: 10.1158/0008-5472.CAN-13-3007 Ó2014 American Association for Cancer Research. 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